s2fieldsofstudy: Biology Medicine

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Cancer-associated fibroblasts and their role in tumor progression

The stromal elements of a malignant tumor can promote cancer progression and metastasis. The structure of the tumor stroma includes connective tissue elements, blood vessels, nerves, and extracellular matrix (ECM). Some of the cellular elements of the tumor stroma are cancer-associated f ibroblasts (CAFs). The origin and function of CAFs have been actively studied over the past thirty years. CAFs produce collagen, the main scaffold protein of the extracellular matrix. Collagen in the tumor stroma stimulates f ibrosis, enhances the rigidity of tumor tissue, and disrupts the transmission of proliferation and differentiation signaling pathways. CAFs control tumor angiogenesis, cell motility, tumor immunogenic properties, and the development of resistance to chemo- and immunotherapy. As a result of metabolic adaptation of rapidly growing tumor tissue to the nutrients and oxygen deprivation, the main type of energy production in cells changes from oxidative phosphorylation to anaerobic glycolysis. These changes lead to sequential molecular alterations, including the induction of specif ied transcriptional factors that result in the CAFs activation. The molecular phenotype of activated CAFs is similar to f ibroblasts activated during inf lammation. In activated CAFs, alpha-smooth muscle actin (α-SMA) is synthetized de novo and various proteases and f ibronectin are produced. Since CAFs are found in all types of carcinomas, these cells are potential targets for the development of new approaches for anticancer therapy. Some CAFs originate from resident f ibroblasts of the organs invaded by the tumor, while others originate from epithelial tumor cells, which are undergoing an epithelial-mesenchymal transition (EMT). To date, many molecular and metabolic inducers of the EMT have been discovered including the transforming growth factor-beta (TGF-β), hypoxia, and inf lammation. This review classif ies modern concepts of molecular markers of CAFs, their functional features, and discusses the stages of epithelial- mesenchymal transition, and the potential of CAFs as a target for antitumor therapy

The biology of cancer-associated f ibroblasts
Modern concept of tumor morphology postulates that solid tumors are formed by epithelial and stromal cells, such as fibroblasts, endothelial cells, and immune cells . Stromal cells with a fibroblast-like phenotype, the so-called cancer-associated fibroblasts (CAFs), in contrast to normal fibroblasts, contain various chromosomal abnormalities, such as duplications, multiple rearrangements, and even the loss of entire chromosomes (Hosein et al., 2010). CAFs control tumor angiogenesis, motility and metastasis of cancer cells, tumor immunogenic properties, and the development of resistance to chemotherapy and immunotherapy (Tripathi et al., 2012;Alkasalias et al., 2018;Nushtaeva et al., 2018).
A meta-analysis of the clinical relevance of the tumor stroma has demonstrated the association of high CAFs content with the advanced stages of tumor progression, as well as with the high risk of local recurrence after tumor resection (Knops et al., 2020).

Heterogeneity of CAFs' percussors cells
In 1995, a heterogeneous origin of CAFs was hypothesized by Rønnov-Jessen and colleagues, who showed that breast cancer CAFs can originate from resident fibroblasts, vascular smooth muscle cells, and pericytes (Rønnov-Jessen et al., 1995). To date, it has been shown that precursors of mesenchymal cells from the red bone marrow, endothelial and epithelial cells, resident fibroblasts of the affected tissue, adipocytes and vascular adventitia cells can be sources of CAFs (Puré, Hingorani, 2018;Yin et al., 2019). For the initiation of the CAFs phenotype in some progenitor cells, additional stimulation with cytokines and growth factors, such as transforming growth factor beta (TGF-β), fibroblast growth factor (FGF), and other signaling molecules is required (Table 1) (Bordignon et al., 2019).
Tumor epithelial cells can undergo transformation into CAFs via the epithelial-mesenchymal transition (EMT) (Fig. 1). EMT is a dynamic process of transdifferentiation of epithelial cells into fibroblast-like cells. The EMT plays an important role not only in cancer, but also in embryogenesis and regeneration. In particular, EMT occurs in embryonic stem cells producing mesoderm and neural crest, and in skin cells during wound healing (Kim et al., 2017). Dynamic changes in cell morphology during EMT are caused by changes in the regulatory genes' expression with production of certain proteins. These proteins are considered as EMT markers (see Fig. 1). Among these markers, the most significant are N-cadherin and vimentin, which are responsible for the rear-rangement of the cytoskeleton and the change in the shape of the cell, as well as the change in cell-to-cell and cell-toextracellular matrix (ECM) interactions (Massagué, 2008;Ye, Weinberg, 2015).
In addition to molecular inducers of EMT, the important role of hypoxic conditions has been shown. Hypoxia activates EMT via the binding of the hypoxia-inducible factor (HIF-1) to the promoters of genes responsible for EMT activation. HIF-1 has been shown to increase the expression of the transcription factors genes of the zinc finger motif family such as ZEB1, Snail and SLUG. Overexpression of these factors is associated with the mesenchymal phenotype and a decrease in the abundance of epithelial cell markers -E-cadherin and type 1 tight junction protein (TJP1 or ZO-1) (Nushtaeva et al., 2019;Tam et al., 2020).
Endothelial cells of tumor vessels can undergo an endothelial-mesenchymal transition (EndMT) and acquire the phenotype and functional features of CAFs with the loss of endothelial cells molecular markers, such as the endothelial cell/platelet adhesion molecule (CD31), and the acquisition of markers specific for mesenchymal cells, such as α-SMA and fibroblast specific protein 1 (FSP-1) (Zeisberg et al., 2007).
An important component of breast cancer stroma are adipose cells, which can transform into tumor-associated adipocytes, and then into CAFs. Such changes are accompanied by an increase in the expression of molecular markers of mesenchymal cells, including, PPARG (receptors induced by peroxisome activators gamma), RUNX-2 (transcription factor containing the Runt type 2 DNA-binding domain), and SOX9 (transcription factor of the HMG family DNA-binding proteins) (Bochet et al., 2013;Liu et al., 2021).
Using the model of prostate cancer, it was shown that mesenchymal stem cells (MSCs) can differentiate into CAFs after the activation of the chemokine receptor type 6 (CXCR6) by its ligand CXCL16. Moreover, the activation of CXCR6 results in the secretion of stromal factor-1 (CXCL12) involved in EMT (Jung et al., 2013). Weber and colleagues also showed that the extracellular structural protein osteopontin (OPN), which plays a key role in bone formation, activates TGF-β gene expression in integrin-dependent MSCs to maintain the phenotype of CAFs in breast cancer. Interestingly, even specialized cells such as Ito cells in the liver, pancreatic stellate cells, and mammary myofibroblasts can acquire the phenotype of CAFs (Weber et al., 2015). These examples illustrate a wide range of cells that, responding to the molecular changes in a tumor, are able to acquire the CAFs' phenotype and, as a consequence, be involved in tumor homeostasis.  Notе. PDGF -platelet derived growth factor; HSF-1 -heat shock factor 1; CLIC4/Arf6 -intracellular chloride channel 4, ADP-ribosylation factor 6; RAS/MAPK -mitogen activated protein kinase; PI3k/AKT -phosphoinositide 3-kinase, alpha serine/threonine protein kinase; TRiC/CCT is a chaperonin containing TCP-1.

Markers of cancer-associated f ibroblasts
The involvement of CAFs in carcinogenesis and tumor progression makes them a potential target for the development of novel therapeutic approaches. A potentially clinically significant marker of CAF is the transmembrane mucin-like protein podoplanin (PDPN) ( Table 2); to date, PDPN has been described as a marker of lymphoid capillary progenitor cells and CAFs in lung cancers. Expression of podoplanin was showed in 54 (30.5 %) out of 177 CAFs' populations studied in the work of Yurugi et al. Interestingly, all podoplanin-positive CAFs correlated with invasiveness of adenocarcinomas, while a podoplanin-negative phenotype was shown only in noninvasive adenocarcinomas (Yurugi et al., 2017). Platelet-derived growth factor receptors α/β are important markers of CAFs. PDGFRα/β belong to the 3rd class of tyrosine kinases and are activated by interaction with the PDGF ligand. PDGFR regulates the organogenesis of various systems during embryogenesis; however, the significance of the PDGFRα and -β receptors activation in tumors is still poorly understood. It has been shown that the expression of the PDGFRβ receptor is increased in the tumor microenvironment cells, where platelet growth factor activates CAFs and, probably, stimulates cancer progression (Anderberg et al., 2009). PDGFRα-positive CAFs have been found in the stroma of melanoma, suggesting that these CAFs originate from resident fibroblasts as a result of their activation (Lynch, Watt, 2018). Serum amyloid A (SAA-1) protein is one of the potential targets of CAFs; its expression and involvement in tumor progression has been shown in CAFs from gastric tumors (Yasukawa et al., 2021).
In the search for specific markers of the tumor stroma cells, among the CAFs of prostate adenocarcinoma, an increased content of the surface protein with a single V-domain of immunoglobulin (CD90), initially found on T cells and neurons, was identified as a specific marker. The high level of CD90 on the cell surface differentiates the tumor-associated stroma and Notе. α-SMA -alpha-smooth muscle actin; PDGFRα/β -platelet-derived growth factor receptors α/β.
"benign" stroma. Since CD90 expression was shown only in tumor associated fibroblasts, this marker is a potential target for therapy (True et al., 2010). Certain CAFs proteins can be prognostic markers of tumor invasiveness. One of these markers is a protein from the family of low molecular weight calcium-binding proteins of the S100 -S100A4 family (Fei et al., 2017). S100 family proteins have both intracellular and extracellular activity due to maintaining the calcium balance and Ca 2+ -dependent processes. S100A4 activates a cascade of reactions associated mainly with the secretion of pro-inflammatory cytokines and the expression of growth factors, extracellular matrix proteins, metalloproteinases, and others. Intracellular activity of S100A4 is of particular interest, and is associated with the enhancement of the invasive capabilities of tumor cells, their escape from apoptosis, and the stem phenotype of the cells (Ambartsumian et al., 2019). During the study of the role of S100A4 in tumor progression, it was shown that suppression of S100A4 decreased tumor growth (Joyce, Pollard, 2009;Grum-Schwensen et al., 2015). The role of stromal cells that secrete S100A4 was shown in the MMTV-PyVmT mouse model with the S100A4 knocked-out gene during orthotopic co-transplantation of CSML100 mouse mammary adenocarcinoma cells and MEF mouse embryonic fibroblasts. MEF cell lines were obtained by spontaneous immortalization of primary embryonic fibroblasts from mouse embryos with the S100A4 + and S100A4phenotypes. Upon co-transplantation of tumor cells and fibroblasts with the S100A4phenotype in syngeneic mice, no metastases were formed, however, upon transplantation of S100A4 + fibroblasts, the metastatic potential of tumor cells returned. S100A4 + fibroblasts were characterized by increased mobility and invasiveness compared to S100A4fibroblasts, as well as the ability to secrete S100A4 into the tumor microenvironment (Grum-Schwensen et al., 2005).
To date, it is clear that the expression of certain CAFs markers does not unambiguously predict the aggressiveness of the tumor. For example, the loss of caveolin-1 in breast cancer CAFs has been shown to be associated with a poor prognosis because the population of these cells stimulates the growth of triple negative (ER-/PR-/HER2-) breast cancer cells (Witkiewicz et al., 2009). In a parallel study, the expression of caveolin-1 in the breast cancer CAFs stimulated the remodeling of the tumor microenvironment, thereby facilitating the invasion of malignant cells and an increased invasiveness level correlated with the metastatic potential of the tumor (Witkiewicz et al., 2009;Goetz et al., 2011). These contradicting re sults indicate the diverse role of caveolin-1 in histologically different tumors. More studies should be made to determine caveolin-1 as a tumor prognostic marker.

Role of cancer-associated f ibroblasts in tumor progression
CAFs-dependent stimulation of the tumor cells proliferation and their invasion is of particular interest in the study of tumor. This interest is primarily due to the fact that even in the precancerous phenotype of epithelial cells, some resident fibroblasts are already transformed into CAFs (Liotta, Kohn, 2001). Fibroblasts from intestinal tumors and polyps were a good model to confirm the contribution of stromal cells to tumor growth and progression. These fibroblasts were shown to stimulate the proliferation of tumor and polyp cells (Mukaida, Sasaki, 2016).
The interaction of tumor epithelial cells with CAFs was analyzed by comparing the histological picture of various types of gastric cancer. In a study by Orimo and Weinberg, it was demonstrated that in the case of diffuse gastric cancer, CAFs and epithelial cells are more closely spaced, while in the intestinal type, CAFs form a stroma-like matrix, due to which tumor epithelial cells retain their glandular structure (Orimo, Weinberg, 2006).
Using the model of heterogeneous 3D spheroids, consisting of breast cancer epithelial cells and fibroblasts, Dang and colleagues showed that CAFs stimulated the migration of tumor cells of basal breast cancer (ER-/PR-/HER2-). Interestingly, this effect was not observed in the models of luminal breast cancer types (ER+/PR+/HER2+, ER+/PR+/HER2-) (Dang et al., 2011). These data are consistent with clinical observations indicating a higher percentage of metastasis in triplenegative breast cancer compared with other types of breast tumors. However, which factors make cells of basal breast cancer sensitive to CAFs stimulation remain undiscovered (Al-Mahmood et al., 2018).
In order to reach the blood and lymphatic streams, tumor cells should pass through the basement membrane (BM), separating them from connective tissue and vessels. Thus, CAFs are able to synthesize metalloproteinases -endopeptidases capable of destroying proteins of all types of the BM extracellular matrix (Gonzalez-Avila et al., 2019). In 2017, Glentis and colleagues revealed a metalloproteinase-independent CAFssupported overcome of BM by tumor cells. They demonstrated the ability of CAFs to stretch BM with the formation of pores, and through these pores, epithelial tumor cells and CAFs can migrate into the bloodstream and form metastases in distant organs. Interestingly, BM regions with low expression of la minin and type IV collagen exhibited the highest tendency for stretching (Glentis et al., 2017). This alternative CAFsde pendent migration pathway explains the ineffectiveness of the metalloproteinase inhibitors application in patients with head and neck tumors.
The paracrine secretion of IL-1α by CAFs in bladder cancer with further activation of the Wnt pathway in tumor cells is the perfect illustration of the pro-carcinogenic role of CAFs (Yang et al., 2021). Moreover, bladder cancer СAFs secreting IL-8 are able to stimulate the secretion of neuropilin-1, which enhances the proliferation of tumor cells and is one of the potential prognostic markers of malignancy (Chen C. et al., 2020). Interestingly, recent studies have shown that neuropilin-1 may be a co-factor in the induction of EMT .
Paracrine stimulation of the epithelial-mesenchymal transition in tumor epithelial cells by CAFs is an important factor contributing to tumor progression. The central mechanism of EMT in the tumor is the TGF-β/Smad pathway activation, induced by TGF-β from stromal fibroblasts (Fig. 2) (Yu et al., 2014). The Smad factor is a transcription factor that controls the expression of EMT genes. Vered and colleagues showed that cells with EMT markers are found in primary foci of squamous cell carcinoma of the tongue as well as in regional lymph nodes metastases. This confirms the importance of CAFs in the induction of metastasis and in the formation of a secondary tumor node (Vered et al., 2010).
The central mechanism of the EMT activation in ovarian cancer is the induction of the CXCR4/Wnt/β-catenin pathway in tumor epithelial cells. CAFs secreting stromal growth factor-1 (SDF-1 or CXCL12) have been shown to be the major players in this process. Moreover, SDF-1 is also interlinked with the resistance of tumor cells to chemotherapeutic agents such as cisplatin .
In 2020, Franzè and colleagues demonstrated the activation of CAFs phenotype in normal fibroblasts from rectal polyps co-cultured with CAFs. They showed that CAFs derived from colorectal tumors can secrete IL-34, which in normal fibroblasts activates the expression of CAFs markers such as α-SMA, vimentin, and fibroblast activating protein (FAP) (Franzè et al., 2020).
Even though most of the described functions of СAFs are associated with the stimulation of tumor progression, some authors also describe СAFs as tumor-suppressing players. For example, the subpopulation of CAFs expressing the melanoma adhesion molecule (CD146) in breast tumors correlated with a retarded cell proliferation in estrogen-dependent types of breast cancer (Brechbuhl et al., 2017). Since CAFs secrete cytokines involved in the recruitment and maturation of macrophages, T-lymphocytes and natural killer cells (IL-10, TGF-β, TNF, IFN-γ and IL-6), they increase the availability of the tumor to immune cells and promote antitumor immune response (Marlow et al., 2008). In the study of oral cancer, it was also shown that CAFs can suppress the proliferation of tumor cells. In particular, the population of CAFs secreting Bone Morphogenetic Protein 4 (BMP4) and expressing α-SMA inhibited the proliferation of cancer stem cells (CSC) (Patel et al., 2018). Using the mice model with a predisposition for the development of pancreatic cancer, Rhim and colleagues exhibited the role of CAFs in cancer progression. They excluded the α-SMA-positive population of CAFs or CAFs with inhibited Hedgehog signaling pathway. These modifications suppress the growth of pancreatic ductal adenocarcinoma. Histological analysis of tumors revealed abnormalities in the vessel's formation (Rhim et al., 2014).

МОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯ / MOLECULAR AND CELL BIOLOGY
These examples demonstrate the tumor-suppressing function of CAFs only in high differentiated cancers with no similarity in undifferentiated ones. It can be assumed that apart from origin and tissue of the affected organ, the function of CAFs is determined by the differentiation stage of cancer cells (Bu et al., 2019).

Isolation of CAFs for research purposes
Cell cultures obtained from patients' tumors after surgery are most often used to study the properties of CAFs. It is necessary to establish new CAFs cell cultures, since in vitro CAFs tend to age rapidly, and the possibilities of their use are limited by early passages (Taddei et al., 2014). Moreover, in commercially available cell collections American Type Culture Collection (ATCC), European Collection of Authenticated Cell Cultures (ECACC), Russian collection of cell cultures, etc. cell lines with the CAFs phenotype are limited. For instance, in ATCC, only one CAFs cell line is available. This cell culture originates from the prostate adenocarcinoma and is modified by the introduction of the telomerase transgene under the control of the constitutive promoter of the polyoma virus SV40 hTERT PF179T CAF. This modification of fibroblasts is aimed at maintaining the proliferative properties of CAFs (Madar et al., 2009).
To obtain cell cultures from tumor tissue, mechanical disaggregation, enzymatic dissociation, chelation and their combination are used. Trypsin and type IV collagenase are most often used to destroy the stroma of tumor tissue. The choice of what technique to use should consider the histological origin of the tissue of interest. When tissue disaggregated, CAFs can represent a small population of cells and obtaining a monoculture requires an additional stage of their separation from the total cell mass. In order to isolate a particular population of CAFs, magnetic separation, or FACS of cells with immunostaining of specific CAFs markers such as FAP or α-SMA are used (Sharon et al., 2013;Huang et al., 2017;Sha et al., 2018). The main difficulty in isolating CAFs lies in adapting protocols for vital staining of intracellular markers such as α-SMA, FAP, and vimentin. Therefore, it is highly desirable to include surface markers such as CD90 in the analysis.

Conclusion
Clear understanding the tumor microenvironment role is crucial for the development of new approaches in cancer diagnostics and treatment. The multifaceted influence of CAFs on tumor progression makes them an important object for the study of carcinogenesis and the development of new antitumor agents. The use of drugs targeted to the components of the tumor microenvironment has not demonstrated efficacy for anti-metalloproteinase compounds and angiogenesis inhibitors as well as T-cell immunity checkpoints inhibitors in some types of cancer (Wang-Gillam, 2019). The heterogeneity of the molecular phenotypes of CAFs can be an important factor in the failure of CAF-targeted cancer treatment. The scientific community should develop more detailed classifications of various subtypes of CAFs considering their involvement in tumor progression.
Thus, a detailed classification of CAFs and a study of the functions of each phenotypic subgroup may provide im-portant knowledge for the development of new methods for the CAF-related treatment and diagnosis of oncological diseases.


s2fieldsofstudy: Biology Psychology

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Animal models to investigate the effects of inflammation on remyelination in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS). In people with MS, impaired remyelination and axonal loss lead to debilitating long-term neurologic deficits. Current MS disease-modifying drugs mainly target peripheral immune cells and have demonstrated little efficacy for neuroprotection or promoting repair. To elucidate the pathological mechanisms and test therapeutic interventions, multiple animal models have been developed to recapitulate specific aspects of MS pathology, particularly the acute inflammatory stage. However, there are few animal models that facilitate the study of remyelination in the presence of inflammation, and none fully replicate the biology of chronic demyelination in MS. In this review, we describe the animal models that have provided insight into the mechanisms underlying demyelination, myelin repair, and potential therapeutic targets for remyelination. We highlight the limitations of studying remyelination in toxin-based demyelination models and discuss the combinatorial models that recapitulate the inflammatory microenvironment, which is now recognized to be a major inhibitor of remyelination mechanisms. These models may be useful in identifying novel therapeutics that promote CNS remyelination in inflammatory diseases such as MS.

Introduction
Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS), characterized by peripheral immune infiltration, demyelination, reactive gliosis, and axonal degeneration (Reich et al., 2018). Multiple disease-modifying drugs have been effective in the treatment of the inflammatory stage of the disease and demonstrated a significant reduction in the formation of new lesions and annualized relapse rate (Bierhansl et al., 2022). However, none of these drugs meaningfully slow the accumulation of disability in progressive MS cases in which there are no superimposed relapses or new MRI lesions. Chronic demyelination renders axons susceptible to neurodegeneration through loss of trophic support, and remyelination is neuroprotective and possibly could restore function. While numerous potential remyelinating compounds have been identified in preclinical research, they have shown limited success in MS clinical trials (Gharagozloo et al., 2022). In MS, chronic inflammation and axonal pathology interfere with remyelination. Both intrinsic defects in oligodendrocyte lineage cells and extrinsic inhibitory cues in the lesion microenvironment negatively affect oligodendrocyte progenitor cells (OPC) differentiation and the generation of new myelin sheaths (Gharagozloo et al., 2022). Nevertheless, the use of animal models is essential for understanding the complex cellular and molecular mechanisms underlying these processes and for developing successful remyelination therapies for MS.
Over the past several decades, various animal models have been used to test novel therapies for MS and study the immunopathological mechanisms. The most commonly used animal model of MS is experimental autoimmune encephalomyelitis (EAE), in which neuroinflammation is the predominant feature observed after immunization with myelin-derived antigens. EAE reflects important aspects of MS immunopathology and has directly led to the development of several effective approved therapies for MS. However, the utility of this model is limited for understanding the remyelination process and identifying strategies for myelin repair due to the unpredictable spinal cord locations of lesions, and the extensive axonal pathology that occurs in this model. Toxin-based models such as cuprizone are commonly used to study demyelination and remyelination in the CNS, but they mostly lack the chronic inflammation that is known to exist in MS plaques. To better recapitulate the complexity of MS pathology, several mouse models have been developed that combine an immune response with toxin-induced demyelination. These combinational models may be useful to identify the molecular factors contributing to remyelination failure and to test novel therapies that promote myelin repair and prevent MS progression.
In this review, we describe the current animal models that have provided insight into the mechanisms by which demyelination and impaired remyelination occur in MS. We highlight the complexities and challenges of studying remyelination, while discussing future perspectives of using animal models to develop novel remyelination therapies for MS. We summarize the current de-and re-myelination models in Table 1 and further discuss the combinational inflammatory models in Figure 1.

Toxin-induced central nervous system demyelination
One robust way to model demyelination and remyelination is via the use of cuprizone induced-demyelination. Cuprizone (biscyclohexanone oxaldihydrazone) is a copper-chelating chemical that is orally administered to rodents to induce oligodendrocyte death, glial cell activation, and demyelination via mitochondrial dysfunction (Matsushima and Morell, 2001). Cuprizone's presumed mechanism of action is through mitochondrial injury driven by the suppression of complex IV of the electron transport chain and oxidative stress due to the increased formation of reactive oxygen species (ROS) and subsequent DNA damage. These mitochondrial-linked impairments reduce the energy supply for the cell, leading to cell starvation, injury, and death (Faizi et al., 2016).
The cuprizone model can be segmented into two stages: CNS demyelination in the corpus callosum and other parts of the cerebrum accompanied by innate immune activation followed by extensive remyelination of the denuded axons upon cessation of cuprizone (Toomey et al., 2021). In the acute stage, demyelination occurs within 4-6 weeks of oral cuprizone exposure (Zhan et al., 2020). Demyelination appears to be directly linked with mature oligodendrocyte death, which starts occurring within 1-2 weeks of toxin exposure. Cuprizone-induced innate immune activation within the CNS follows as reactive microglia and neurotoxic astrocyte phenotypes become more prominent.
Following the demyelination that occurs upon acute cuprizone ingestion, oligodendrocyte precursor cells (OPCs) begin to proliferate and migrate to the areas of demyelination, even while the animals are ingesting cuprizone since these non-myelinating cells are much less metabolically active and can survive the mitochondrial toxicity. However, successful remyelination is dependent on cessation of cuprizone to allow OPCs to differentiate into mature myelinating oligodendrocytes. OPCs have been shown to be the major source of remyelination in this model. In the acute cuprizone model, the toxin is discontinued at 3-6 weeks allowing remyelination to occur in the ensuing 2-4 weeks. Cuprizone diet triggered extensive corpus callosum (CC) demyelination, reduced axon conduction, and resulted in loss of axon structural integrity including nodes of Ranvier. Indeed, there is a window of opportunity for axonal remyelination following cuprizone withdrawal. Demyelination longer than 1.5 weeks can cause axonal injury that limits successful remyelination of axons to normal levels by oligodendrocytes (Crawford et al., 2009). In the chronic cuprizone model, the toxin is administered for 8-12 weeks, and axonal damages ensues limiting the potential for complete remyelination. The corpus callosum also thins in the chronic model as a sequela of axonal injury that occurs in MS. Although acute cuprizone treatment allows for more rapid remyelination, chronic cuprizone treatment still results in myelin repair of the surviving axons (Kipp et al., 2009;Lindner et al., 2009). Other toxin-based methods to reproduce demyelination and remyelination as may occur in MS pathology include lysolecithin (lysophosphatidylcholine-LPC) and diphtheria toxin administration. LPC non-specifically binds to myelin and disrupts the lipid architecture. LPC has been shown to rapidly integrate into the membranes of cells and similarly cause deformation and subsequent permeabilization of these cells (Plemel et al., 2017). In the diphtheria toxin (DT) model, the diphtheria fragment A gene (DTA) can be conditionally expressed in oligodendrocytes, leading to oligodendrocyte death and CNS demyelination (Popko, 2010). Alternatively, transgenic mice have been made with the diphtheria toxin receptor expressed under an oligodendrocyte specific promoter and upon deliver of diphtheria toxin rapid demyelination ensues (Brockschnieder et al., 2006;Popko, 2010;Traka et al., 2010;Pohl et al., 2011;Plemel et al., 2017).
There are many beneficial aspects of utilizing toxins to model the myelination processes that occur in MS. Acute and chronic demyelination with associated CNS atrophy can be modeled using cuprizone, LPC, and diphtheria toxin and have similarities to aspects of human MS pathology (Peterson et al., 2001;Buch et al., 2005;Kutzelnigg et al., 2005;Traka et al., 2010;Goldberg et al., 2015;Wagenknecht et al., 2016;Pang et al., 2022). Axonal damage in demyelinated lesions along with minor peripheral immune infiltration are similarly aligned with progressive MS (Höflich et al., 2016;Singh et al., 2017;Machado-Santos et al., 2018). Axons are more preserved in the LPC model as opposed to prolonged cuprizone exposure (Kirk et al., 2003;Berghoff et al., 2017;Filippi et al., 2018;Chrzanowski et al., 2019).
Despite the advantages of using these toxins to induce demyelinating pathology, there are various limitations to consider when attempting to model MS. Given that cuprizone is a toxin that poisons mitochondria activity and induces a degenerative loss in the CNS, the adaptive immune system characteristics of MS are lacking (Baxi et al., 2015). Indeed, cuprizone-induced demyelination is not lymphocyte-dependent and occurs even in lymphocyte-deficient recombination activating gene (Rag) knockout mice (Hiremath et al., 2008). Additionally, susceptibility to cuprizone pathology can vary based on animal weight and genetic background, cuprizone consumption and formulation means of cuprizone delivery, and animal age (Carey and Freeman, 1983;Zatta et al., 2005;FIGURE 1 Schematic overview of inflammatory demyelination models, showing how inflammation contributes to de-and re-myelination following cuprizone intoxication. ATCup, Adoptive Transfer Cuprizone; CAE, Cuprizone Autoimmune Encephalitis; CFA, Complete Freund's Adjuvant; DOX, Doxycyclin; EAE, Experimental Autoimmune Encephalomyelitis; MOG, Myelin oligodendrocyte glycoprotein; OPC, Oligodendrocyte precursor cells; PTX, Pertussis toxin. Irvine and Blakemore, 2006;Taylor et al., 2010;Steelman et al., 2012;Hochstrasser et al., 2017;Yu et al., 2017;Leopold et al., 2019). In the following sections, we will focus on inflammatory immune response in toxin-based demyelination models and review several models that have incorporated an induced immune response to further our understanding of potential mechanisms of remyelination failure in MS.

Toxin-based demyelination and inflammatory immune response
Many studies show the autoimmune origin of MS, in which autoreactive T cells drive multifocal and heterogeneous inflammatory demyelination (Rodríguez Murúa et al., 2022). Some findings suggest that brain-intrinsic pathologies such as primary apoptosis of oligodendrocytes trigger the recruitment of peripheral immune cells into the CNS. Based on these observations, two models have been proposed to describe the etiology of MS, the "outside-in" and the "inside-out" models (Titus et al., 2020). The outside-in paradigm suggests that immune cells are primarily activated in the periphery and attack the CNS, while inside-out implicates a primary CNS cytotoxic process such as oligodendrocyte death that triggers secondary autoimmune reactions against myelin antigens (Stys et al., 2012). The cuprizone model is considered an appropriate approach to test the "inside-out" hypothesis since cuprizone toxicity is lymphocyte-independent and primarily caused by oxidative stress and mitochondrial dysfunction of oligodendrocytes (recently reviewed by Zirngibl et al., 2022).
Some studies tested whether cuprizone-induced demyelination could activate the adaptive immune response, causing T cell infiltration into the CNS. However, there are controversial observations regarding the impact of cuprizone on inflammatory demyelination models. Earlier studies suggest that acute cuprizone intoxication does not induce a blood-brain barrier (BBB) permeability (Bakker and Ludwin, 1987;Kondo et al., 1987;McMahon et al., 2002) and T cell infiltration to the CNS (Matsushima and Morell, 2001;Remington et al., 2007;Hiremath et al., 2008). Even after pertussis toxin administration to breach the BBB, T cell infiltration is not detectable in the CNS (Sen et al., 2019). In contrast, other studies show that BBB integrity is compromised in mice and precedes the demyelination and glial activation observed in cuprizone treatment (Berghoff et al., 2017;Shelestak et al., 2020). Cuprizone-induced demyelination may trigger the recruitment of specific peripheral immune cells, particularly CD8 + lymphocytes, which predominantly display a cytotoxic phenotype with high proliferation rates and cytotoxic granule expression. This finding indicates an antigenic and proinflammatory milieu in the CNS of cuprizone-treated mice (Kaddatz et al., 2021).
It is unclear whether infiltrating T cells are activated against myelin antigens in the cuprizone model. Although myelin is found in the cervical lymph nodes following cuprizonetreatment, it seems not to be sufficient to trigger an immunemediated CNS inflammation . It is shown that the numbers of inflammatory effector T cells (Th 1 and Th 17 cells) are unchanged in peripheral lymphoid organs and adoptive transfer of splenocytes from cuprizone-treated animals to recipient mice does not result in CNS autoimmunity . Similar results were reported by Locatelli et al. using oDTR model, in which mature oligodendrocyte ablation is induced by the administration of diphtheria toxin. Despite the presence of myelin antigens in the CNS-draining lymph nodes, T cells are not primed against myelin antigens even after pertussis toxin administration as an enhancer of CNS inflammation (Locatelli et al., 2012). These findings suggest that oligodendrocyte death would not be enough to trigger an adaptive immune response and trafficking of the peripheral immune cells into the CNS. Interestingly, another study using a diphtheria toxin (DTA) mouse model suggests that myelin degeneration can cause immune-mediated neuroinflammation and tissue damage at the later stage (Traka et al., 2016). Oligodendrocyte ablation using diphtheria toxin in Plp1-CreER(T);ROSA26-eGFP-DTA (DTA) mouse model induces a fatal late-onset disease (approximately 30 weeks after recovering from oligodendrocyte loss and demyelination), associated with extensive demyelination, T cell infiltration to the CNS, and axonal loss. Myelin oligodendrocyte glycoprotein (MOG)specific T cells were found in lymphoid organs that could induce EAE in naive recipients upon adoptive transfer. Taken together, this study indicates that oligodendrocyte death in the chronic stage would be sufficient to trigger an adaptive autoimmune response against myelin (Traka et al., 2016).
T cell recruitment was also reported when cuprizone intoxication was combined with a non-specific immune stimulus such as Complete Freund's Adjuvant (CFA) and pertussis toxin (Caprariello et al., 2018). In this model, known as cuprizone autoimmune encephalitis (CAE), mild cuprizone intoxication (only 2 weeks) is followed by a non-specific activation of immune cells using CFA and pertussis toxin (Caprariello et al., 2018). Substantial astrogliosis and infiltration of leukocytes including T cells and macrophages are observed in CAE lesions, indicating secondary inflammatory demyelination like active MS lesions. This finding suggests that subclinical myelin injury may trigger the neuroinflammation that subsequently results in oligodendrocyte loss and demyelination. This finding provides a rationale for the inside-out model of MS pathogenesis.

Demyelination combined with central nervous system autoimmunity
Toxin-based models largely lack the inflammatory microenvironment that exists in the MS lesions. In the EAE model, the infiltration of inflammatory immune cells causes severe demyelination and axonal degeneration, limiting the ability of the model to test the novel remyelination strategies (Soulika et al., 2009). In the cuprizone model, however, a robust demyelination and remyelination occur in predictable time frame and regions of the CNS but the endogenous repair is very fast (within 2-3 weeks), providing a limited period to test the drugs that accelerate the spontaneous repair process (Vega-Riquer et al., 2019). For these reasons, combinational models have been developed to enable the study of remyelination in the context of ongoing inflammation (Baxi et al., 2015;Rüther et al., 2017).
Several studies reported that cuprizone treatment can modulate the course and the severity of EAE, induced by active immunization with myelin antigens (MOG or PLP) or Theiler's murine encephalomyelitis virus (TMEV) (Emerson et al., 2001;Maña et al., 2009;Herder et al., 2012). It is shown that cuprizone intoxication generates adaptive immune response against several myelin components that are tolerogenic and/or decrease CD4+/CD8+ ratio, thereby impedes progression of EAE (Emerson et al., 2001;Maña et al., 2009). Cuprizone has also shown to be immunosuppressive and causes the atrophy of peripheral lymphoid organs such as the thymus or spleen (Solti et al., 2015;Sen et al., 2019). In contrast, there are studies that identified a comparable severity of EAE clinical symptoms in mice with or without cuprizone intoxication.
Recent studies describe a model combining cuprizone demyelination with the classic active EAE (Cup/EAE). In this model, inflammatory lesions are detected at various forebrain areas, including white matter, cortical, and subcortical gray matter Rüther et al., 2017). The neurodegenerative process was induced by cuprizone feeding of adult mice for 3 weeks, followed by a period on normal chow . Subsequent immunization with MOG 35-55 peptide induces myelin autoreactive T cells in the periphery, resulted in massive recruitment of immune cell including encephalitogenic T cells together with monocytes into the affected forebrain. In addition, mice fed cuprizone before adoptive T-cell transfer showed a higher number of perivascular infiltrates compared with animals without previous cuprizone feeding Rüther et al., 2017). This study clearly illustrates the significance of brain-intrinsic degenerative cascades for immune cell recruitment and lesion formation. Similarly, Locatelli et al. tested whether oligodendrocyte death interfered with EAE development using oDTR mice. The EAE clinical scores and disease incidence were found comparable in all of the mice and the level of activated inflammatory infiltrates were similar at the onset (Locatelli et al., 2012). These findings suggest that cuprizone itself but not oligodendrocyte death ameliorates the course of EAE.
To overcome the limitation of cuprizone in the active EAE model, our group developed a adoptive transfer cuprizone model (ATCup) by combining cuprizone and adoptive transfer of activated myelin-specific CD4+ T cells. Mice are fed cuprizone for 4 weeks to induce demyelination and then adoptively transferred with myelin reactive CD4+ T cells that have been polarized to the Th17 profile in vitro (Baxi et al., 2015). We found IFNγ/IL-17 dual producing CD4 + T cells in the brain of cuprizone-fed mice following adoptive transfer, resulting in the significant inhibition of spontaneous remyelination. While in this system, the ensuing CNS inflammatory response inhibits endogenous remyelination, axons are preserved (unlike in the spinal cord of EAE animals). This preservation of axons allows for the capacity to examine the efficacy of drugs on OPC differentiation in a system where remyelination typically fails, similar to what may happen in MS plaques.
To better understand the mechanisms by which myelin reactive T cells inhibit remyelination, we performed AT-Cup in a lineage tracing OPC reporter mice (PDGFRα-CreER x Rosa26-YFP), in which we fate mapped OPCs in the regular cuprizone model (Baxi et al., 2017). In this model, OPCspecific Cre recombination occurs upon administration of 4hydroxytamoxifen (4-HT) during the 3rd week of cuprizone and OPCs become positive for yellow fluorescent protein (YFP), a heritable trait that will remain even as they divide or differentiate into oligodendrocytes. We assessed the differentiation of YFP + OPCs during remyelination and after adoptive transfer of activated T cells and found that inflammatory T cells inhibit the differentiation of OPCs into mature oligodendrocytes in the corpus callosum (Kirby et al., 2019).
Taken together, the combinational inflammatory remyelination models, Cup/EAE and ATCup, could be useful for testing remyelination therapies for MS in the context of active inflammation. In this setting, T cells produce inflammatory cytokines (IFNγ and IL-17) that suppress spontaneous remyelination. In addition, microglia and astrocytes respond to T cell cytokines and are polarized toward a reactive phenotype that further suppress remyelination (Gharagozloo et al., 2022;Kunkl et al., 2022).

Demyelination in the genetic models of inflammatory central nervous system
One of the inflammatory factors that has been implicated in impaired remyelination in MS lesion is IFNγ, mainly produced by activated T cells and natural killer cells. To examine the molecular mechanisms of IFNγ-induced oligodendrocyte injury, the Popko lab used transgenic mice that allow temporally controlled expression of IFNγ in the CNS using the tetracycline controllable system (Lin et al., 2006). These animals exploit the transcriptional regulatory region of the glial fibrillary acidic protein (GFAP) gene to drive a tetracycline-controlled transcriptional activation (tTa) system's expression in astrocytes as an ectopic source of IFNγ in the CNS. GFAP/tTA mice were crossed with TRE/IFNγ mice to produce GFAP/tTA;TRE/IFNγ double transgenic animals (Lin et al., 2004), in which mice begin expressing IFNγ in the forebrain after 2 weeks of doxycycline removal. It is shown that ectopic expression of IFNγ in the CNS after cuprizone-induced demyelinating results in reduced oligodendroglia maturation and impaired remyelination (Lin et al., 2006).
Mechanistically, IFNγ can directly affect oligodendrocytes, inducing endoplasmic reticulum (ER) stress response that leads to the apoptotic cell death (Lin et al., 2006). Moreover, we found that IFNγ polarizes OPC to an immune phenotype (iOPCs), in which OPC express immunoproteasome subunit and MHC class I, allowing cross-presentation of antigen to cytotoxic CD8 T cells and resulting in OPC death (Kirby et al., 2019). IFNγ may also have a secondary effect on oligodendrocytes, possibly through the activation of microglia and polarizing astrocytes toward a reactive/neurotoxic phenotype (Liddelow et al., 2017). Activated microglia produce inflammatory mediators including interleukin-1α (IL-1α), tumor necrosis factor (TNF) and complement component 1q (C1q) that generate reactive astrocytes. Toxic lipoparticles containing saturated lipids (APOE and APOJ) are secreted by neurotoxic astrocytes that mediate oligodendrocytes death (Guttenplan et al., 2021). We have recently examined the functional effect of neurotoxic astrocyte conditioned media (ACM) on OPC using a myelin basic protein (MBP) reporter line. Our data show that reactive astrocytes are not cytotoxic to the OPCs but robustly inhibit their maturation to oligodendrocytes, thereby limiting their remyelination capacity (Smith et al., 2022).

Concluding remarks
There are many model systems to capture specific facets of MS pathophysiology. However, it is exceedingly difficult to recapitulate most features of MS within the same model given the multifaceted, complex neuroimmunological and neurodegenerative pathology that occurs. Nevertheless, several MS therapeutics have been clinically developed in the EAE model that have shown proven benefit in MS. However, remyelination therapy for MS remains challenging since many remyelinating drugs have shown promise preclinically but have had minimal benefit in clinical trials. The translational gap between animal models and clinical trials can be bridged by developing combinational models that better recapitulate MS pathology, incorporating the influence of inflammatory molecules that inhibit remyelination within the lesion. There is much excitement within the field as it progresses with the use of combinatorial model systems that incorporate the main hallmarks of MS such as remyelination and autoimmune immunopathology, innate immune -mediated inflammation, and neurodegeneration. Understanding the complex mechanisms underlying impaired remyelination in MS will provide us with more targeted and effective therapeutic approaches that promote remyelination and halt MS disease progression.


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CircEPSTI1 Promotes the Proliferation of HER2-Positive Breast Cancer Cells via circEPSTI1/miR-145/ERBB3 Axis

Breast cancer is the most common type of cancer worldwide. There are great challenges in the prevention and treatment of breast cancer. In this study, we explored the molecular and biological mechanisms of circular RNA circEPSTI1 (has_circ_0000479) in the regulation of HER2-positive breast cancer cells. The expression of CircEPSTI1, microRNA miR-145, and ERBB3 in HER2-positive breast cancer cells was evaluated by qRT-PCR and western blot assays. Cell proliferation was assessed by CCK-8. Wound-healing and transwell migration assays were performed to evaluate cell migration. A transwell invasion assay was performed to detect cell invasion. The interaction of miR-145, circEPSTI1, and ERBB3 was confirmed bydual-luciferase reporter and RIP assays. CircEPSTI1 was upregulated in the HER2-positive breast cancer tissues and cells. Knockdown of circEPSTI1 inhibited SKBR3 and BT474 cell proliferation, migration, and invasion. Mechanistically, circEPSTI1 directly targeted miR-145, and miR-145 was a downstream mediator of circEPSTI1 in modulating the proliferation, migration, and invasion of SKBR3 and BT474 cells. ERBB3 was identified as a direct and functional target of miR-145 in HER2-positive breast cancer cells. Our findings demonstrate that circEPSTI1, an overexpressed circRNA in HER2-positive breast cancer, promotes the proliferation, migration, and invasion of SKBR3 and BT474 cells through the miR-145/ERBB3 axis.

Introduction
Breast cancer is one of the most common cancers worldwide, accounting for about 30% of female cancers, and has a mortality-to-incidence ratio of 15% [1]. Globally, the incidence of breast cancer ranges between 27 in 100,000 (for Africa and East Asia) and 97 in 100,000 (for North America) [2]. Human epidermal growth factor receptor 2 (HER2/ ERBB2) positive breast cancer accounts for 20-25% of all breast cancers. Although the development of HER2-directed therapy has revolutionized the mode and outcome of treatment in patients, the high toll of HER2-positive breast cancer deaths persists [3,4]. Currently, the development of drug resistance, unfavorable side effects of TKIs, and tumor metastasis are big challenges in the management of breast cancer [3].
CircRNA is a type of noncoding RNA that forms a covalently closed continuous loop [5]. Based on this feature, circRNAs exhibit a variety of functions, and many of them were discovered in recent years [6]. CircRNAs modulate target gene expression by binding to RNA-binding proteins [7]. In addition, it has been shown that circRNA can sponge miRNAs to control the function of miRNAs [8]. A number of studies have found that altered circRNA expression in various human cancers and circRNA play crucial roles in tumorigenesis.
MicroRNAs (miRNAs) also belong to noncoding RNA that play a central role in cell proliferation, differentiation, and survival by targeting complementary mRNAs, contributing to mRNA translational inhibition or degradation [9]. Wei et al. [10] found that miR-422a binds to the 3′-UTR of MAPK1 and activates Raf/MEK/ERK signaling pathway to promote colorectal cancer cell proliferation. Ferlay et al. confirmed that miR-21 affects the p53 signaling pathway [11], a well-known tumor suppressive gene [12,13]. Besides, as miRNAs are associated with cancer metastasis, a new clinical diagnostic target based on miRNA signatures is being studied to identify subtypes of breast cancer and predict metastasis or treatment resistance [14][15][16]. miRNAs are capable of acting as biomarkers due to their stability and the ability to resist repeated freezing and thawing [17].
ERBB3 is one of the ErbB family and plays a vital role in the development and progression of tumors [18]. Because of the success of targeting EGFR and ErbB2, targeting ERBB3 is becoming increasingly interesting. A similar strategy can be used to target the latter, as all family members have great similarities in structure. ERBB3 plays an important role in several compensatory processes of tumorigenesis and the emergence of traditional cancer drugs. At the moment, there are no approved anti-ERBB3 therapies in clinical practice, whereas many drugs are at different stages of clinical development [18].
Here, we found that circEPSTI1 was upregulated in 20 paired HER2-positive breast cancer specimens compared to their adjacent normal tissues. Based on the significant differential expression of circEPSTI1, we were supposed to explore its role in HER2-positive breast cancer. Knockdown of circEPSTI1 observably inhibited cell proliferation, migration, and invasion. Furthermore, circEPSTI1 could bind to miR-145 and regulate the expression of ERBB3.
us, circEPSTI1 could be used as a novel biomarker and potential therapeutic target for breast cancer.

Ethical Standards.
is study was approved by the Ethics Committee of Sun Yat-Sen University Cancer Center Health Authority (GZR2017-163). All the procedures were performed strictly in accordance with the ethical standards of the Declaration of Helsinki (as revised in 2013). Patient consent was obtained before the study commenced.

Cell
Culture and Transfection. SKBR3 and BT474 cells were obtained from the American Type Culture Collection (ATCC, VA, USA). Cells were cultured in DMEM media containing 10% FBS (Gibco, USA, Cat.# 16140071) and 1% penicillin-streptomycin (Gibco) at 37°C in a 5% CO 2 incubator. After resuscitation from frozen aliquots, all cells were passaged in the laboratory for less than 6 months and were verified by DNA fingerprinting every 6 months. All si-RNAs, plasmids, and viruses used in this study were obtained from GenePharma (Shanghai, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used for cell transfection. 48 hours and 72 hours after the transfection, cells were collected for qRT-PCR and western blotting study.

Transwell Migration and Invasion Assay.
During the migration assay, we seeded transfected SKBR3 or BT474 cells in the top chamber with a no-serum medium and added a medium with 20% FBS into the lower chamber. During the invasion assay, we covered the transwell inserts (Fisher Scientific, MA, USA) with Matrigel (BD, Franklin Lakes, USA), then seeded transfected SKBR3 or BT474 cells in the top chamber with no-serum medium, and added medium with 20% FBS into the lower chamber. 24 hours after incubation, cells on the upper surface of the transwell chamber were gently removed with a cotton swab, and cells on the lower surface were fixed with methanol. en the cells were stained with 0.5% crystal violet (Solarbio), imaged, and counted.

2.7.
Wound-Healing Assay. In 6-well cell culture dishes, 1 × 10 5 transfected SKBR3 cells were seeded in each well. When the cells achieved about 95% confluence, a sterile 200-μL pipette tip was used to create a scratch across the cell monolayer. Images were photographed at 0 and 24 h. Wound closure analysis was performed using ImageJ software (National Institute of Health, Bethesda, MD, USA).

Luciferase Reporter Assay.
By cloning fragments of the circEPSTI1 or ERBB3 3′ UTR that contain the predicted wild-type or mutant binding sequences of miR-145 into the pmirGLO vector (Promega Corporation, Fitchburg, USA), we were able to detect both wild-type and mutant binding sequences of miR-145. Luciferase reporter vectors circEP-STI1 WT, circEPSTI1 MUT, ERBB3 3′ UTR WT, and ERBB3 3′ UTR MUT were formed. SKBR3 or BT474 cells were transfected with the indicated vector and miR-ctr or miR-145. Using the Dual-Luciferase Reporter Assay Kit (Promega), the luciferase activity was examined 48 hours later.
2.9. RNA Immunoprecipitation Assay. SKBR3 or BT474 cells were planted in 6-well plates and co-transfected when proliferation was 60%-70% with strict requirements. After 48 hours, the Magna RIP Kit (Millipore) was used to perform the RIP assay. An RNA immunoprecipitation assay was performed with an anti-Ago2 antibody (Millipore) for the RIP assay on Ago2.

Statistical
Analysis. SPSS 25.0 software was used for statistical analysis. e t-tests and χ 2 tests were performed for comparisons between groups. Unless otherwise noted, the mean ± SEM of triplicate independent experiments are used to present data. P < 0.05 was considered statistically significant.

CircEPSTI1 Is Increased in HER2-Positive Breast Cancer Cells and Tissues.
e first step was to conduct qRT-PCR in order to characterize the level of circEPSTI1 in HER2positive breast cancer tissues. e result demonstrated that HER2-positive breast cancer tissues had a significantly higher circEPSTI1 expression compared with nearby normal tissues (Figure 1(a)). In addition, circEPSTI1 expression was detected to be increased in SKBR3 and BT474 cells compared with MCF-10A cells (Figure 1(b)). CircRNAs showed an apparent resistance to the degradation mediated by exonuclease. RNase R was employed to treat the total RNA from the HER2-positive breast cancer cell lines. e result confirmed the circular structure of circEPSTI1 by RNase R digestion experiments in SKBR3 and BT474 cells (Figure 1(c)). e results of the subcellular fraction assay showed that circEPSTI1 was mostly enriched in the cytoplasm of SKBR3 and BT474 cells (Figure 1(d)).

CircEPSTI1 Promotes the Proliferation, Migration, and
Invasion of HER2-Positive Breast Cancer Cells. Based on the upregulation of circEPSTI1 in HER2-positive breast cancer, si-circEPSTI1 was transfected to knock down circEPSTI1 to explore its biological function in HER2-positive breast cancer SKBR3 and BT474 cells. e transfection was successfully performed (Figure 2(a)).
e CCK-8 assay indicated that circEPSTI1 downregulation significantly weakened the proliferation of the cells (Figure 2(b)). e Transwell migration and invasion assay confirmed that circEPSTI1 knockdown strongly hindered the migration and invasion processes of these cells (Figures 2(c) and 2(d)). Also, the wound-healing assay confirmed that knockdown of circEPSTI1 obviously suppresses the migration of SKBR3 cells (Figure 2(e)). ese results indicated that circEPSTI1 knockdown suppressed the proliferation, migration, and invasion of HER2-positive breast cancer cells.

CircEPSTI1 Targets miR-145 to Facilitate HER2-Positive
Breast Cancer Progression. As we had found, circEPSTI1 was mainly distributed in the cytoplasm (Figure 1(d)), suggesting that circEPSTI1 might function as a miRNA sponge to adsorb miRNAs. Hence, we investigated the possible interactions between circRNA and miRNA in the Circular RNA Interaction Group (https://circinteractome.nia.nih. gov/index.html) and located complementary sites for miR-145 in the circEPSTI1 sequence (Figure 3(a)). In addition, the expression of miR-145 was obviously decreased in SKBR3 and BT474 cells (Figure 3(b)). en a luciferase assay was performed to explore whether miR-145 could bind to circEPSTI1. Luciferase intensity receded when cotransfected with the luciferase reporter gene and miR-145 mimic (Figure 3(c)). To further confirm the direct binding of circEPSTI1 and miR-145, we performed an RIP assay based on MS2bp-MS2bs. e RIP assay showed that miR-145 was mostly enriched in the MS2bs-circEPSTI1 group, indicating a specific interaction between circEPSTI1 and miR-145 ( Figure 3(d)). According to the above results, we further explored whether circESPTI1 modulated the proliferation, migration, and invasion of HER2-positive breast cancer cells by directly targeting miR-145. SKBR3 and BT474 cells were divided into 4 groups: si-ctr, si-circESPTI1, si-circESP-TI1 + anti-miR-ctr and si-circESPTI1 + anti-miR-145. By cell proliferation experiment (Figure 3(e)), the proliferative capacity, weakened by circESPTI1 knockdown, could be restored by co-transfected miR-145 inhibitor. Transwell experiments showed that the migration and invasion ability, suppressed by circESPTI1 knockdown, was restored by cotransfected miR-145 inhibitor in SKBR3 cells (Figure 3(f )).
ese results suggest that circESPTI1 regulates the proliferation, migration, and invasion of HER2-positive breast cancer cells as a miR-145 sponge, promoting breast cancer progression. Overall, we confirmed that circEPSTI1 could interact with miR-145 and act as a decoy for miR-145.

CircEPSTI1
Regulates ERBB3 as miR-145 Sponge in HER2-Positive Breast Cancer Cells. To explore whether circEPSTI1 decoyed miR-145 to influence the expression of target genes, ENCORI (https://starbase.sysu.edu.cn/) was used to predict the target genes of miR-145 and ERBB3 was identified (Figure 4(a)). Luciferase activity markedly decreased with co-transfected miR-145 mimics by luciferase Journal of Oncology 3 reporter assay (Figure 4(b)). Furthermore, we found that the expression of ERBB3 was inhibited by miR-145 (Figures 4(c) and 4(d)), suggesting that ERBB3 was a direct target of miR-145. CircEPSTI1, ERBB3, and miR-145 were mainly enriched to Ago2 by RIP assay on Ago2 (Figure 4(e)), suggesting that circEPSTI1 and ERBB3 are recruited to an Ago2-related RISC where they interact with miR-145. Additionally, knockdown of circEPSTI1 significantly decreased the enrichment of Ago2 to circEPSTI1 while increasing the enrichment of Ago2 to ERBB3 (Figure 4(f )), confirming that circEPSTI1 could bind to miRNAs and compete with ERBB3 as a ceRNA. en, we investigated the expression of ERBB3 after knocking down circEPSTI1 and found it was inhibited, yet inhibiting miR-145 could reverse the repression (Figure 4(g)). ese findings indicate that circEPSTI1 could act as a miR-145 sponge to ERBB3 expression via the ceRNA mechanism.
To verify whether miR-145 directly acted on ERBB3 to affect HER2-positive breast cancer cells, we divided SKBR3 and BT474 cells into 4 groups: si-ctr, si-circESPTI1, si-circESPTI1 + ERBB3-ctr, and si-circESPTI1 + ERBB3. CCK-8 assay was performed and showed that the suppressing proliferation caused by knocking down circEPSTI1 was basically recovered after co-transfection of ERBB3, contracting to si-circESPTI1 transfected in SKBR3 and BT474 cells (Figure 4(h)). Furthermore, transwell experiments showed that the weakening migration and invasion ability of cells caused by knocking down circEPSTI1 could be recovered after co-transfection of ERBB3 in SKBR3 cells (Figure 4(i)). ese results indicated that miR-145 decreased ERBB3 transcription and translation, and circEPSTI1 promoted her2-positive breast cancer progression by regulating ERBB3 as a miR-145 sponge.

Discussion
Studies on CircRNAs have brought about many interesting findings, indicating that circRNAs play very important roles in the occurrence and development of various tumors [9,19]. CircRNA is a type of noncoding RNA, although recent studies have shown that circRNA also has the potential to code for proteins [20]. Due to their special molecular structure, circRNAs exhibit outstanding stability in cells [21], and they are selective and specific in tissue and organ expression [22]. It has been suggested that circRNA expression is related to the developmental stage or aging  Journal of Oncology upregulated circRNA. It significantly reduces the accumulation of various chemotherapeutic drugs in monastolresistant cell lines [26]. In addition, circRNA may be involved in modulating immune responses. Studies have shown that circCDR1 plays a key role in immune and stromal cell invasion of breast cancer, especially CD8+ T cells, activating immune-related cells such as NK cells, M2 macrophages, and endothelial cells [27]. According to numerous studies, circRNAs can be used as a cancer marker for the diagnosis and prognosis of tumors. Abundant studies have explored the function of miR-145 and found that miR-145 held many functions and had different effects on tumor. miR-145 overexpression increased chemotherapeutic efficiency by inducing intracellular doxorubicin accumulation by suppressing MRP1 in breast cancer [28]. Sheng et al. [29]found that miR-145 had an inhibitory effect on colorectal cancer, reducing cell migration and invasion via suppressing P21-activated kinase 4 (PAK4). miR-145 could interact with circITCH to regulate its target gene RASA, promoting the progression of ovarian cancer [30].miR-145 regulates the SOX2-Wnt/ β-catenin axis to enhance chemosensitivity to demethoxycurcumin in gliomas [31]. Kou et al. [32] found that miR-145 inhibited tumor invasion via targeting PAK1 in bladder cancer cells and enhanced EMT through phosphorylating Snail. ese findings indicate that miR-145 has a strong effect on tumor progression. Upregulated ErbB3 plays a vital role in primary and acquired resistance against ErbB2 drugs, chemotherapy drugs, tyrosine kinase inhibitors (TKIs), and hormone therapy. Numerous studies have found that ErbB3 is overexpressed in breast, ovarian, prostate, colon, pancreatic, gastric, urinary, oral, and lung cancers [33]. In patients diagnosed with estrogen receptor (ER)-positive breast cancer, erbB3 upregulation occurs after treatment with fulvestrant, activating the MAPK/ERK pathway and leading to resistance to antiestrogen therapy [34]. In ERbB2 and ERpositive breast cancer patients, ErbB3 overexpression by ErbB2 manufacturing heterodimers, and activating the PI3K/AKT3 signaling pathway leads to tamoxifen resistance, thereby restraining forkhead box O3 (FOXO3a) transcription factors. Eventually, the nuclear estrogen receptor ERα is downregulated [35].
In HER2-positive breast cancer, we focused on cir-cEPSTI1, revealed the role of miR-145 as its downstream molecule, and verified that ERBB3 is regulated by miR-145 and has significant changes in mRNA and protein levels, which provides a basis for understanding the mechanism of the occurrence and development of HER2-positive breast cancer, and provides the therapeutic possibility of tumor drug resistance, recurrence, and metastasis. ere were some limitations to this study. We only demonstrated that circEPSTI1 is a miRNA sponge. A large number of studies have confirmed that circRNAs have other roles such as communication with RNA-binding proteins (RBPs) [34], translation of proteins or peptides [36], and transcription regulation [37] that eventually produce a variety of different biological effects involved in the regulation of tumor cells, tumor microenvironment, angiogenesis, tumor immunity, and more [38]. In this study, we have elucidated the ability of circRNA as a miRNA sponge to promote the proliferation, migration, and invasion of HER2positive breast cancer cells via targeting ERBB3. We have found one of the mechanisms of malignant behavior of HER2-positive breast cancer. However, the process involving circRNAs is still unclear. At present, there are a considerable number of ERBB3 targeted drugs in clinical and preclinical areas, such as Patritumab, Elgemtumab, Seribantumab, REGN1400, GSK2849330, Anti-NRG antibodies, and others [33]. Combined with their pharmacological mechanisms, it is of great significance to explore the potential role of circRNAs in guiding the treatment of HER2-positive breast cancer, reversing drug resistance, and improving prognosis. In addition, in biological basic research, it is best to combine experiments in vivo and related breast cancer clinical data to further illustrate the role and effect of circEPSTI1 in the body environment, which is conducive to the construction of a possible network of circRNAs as a biomarker in breast cancer occurrence, development, prognosis, medication guidance and other aspects of an application.

Conclusions
In this study, we elucidated that circEPSTI1 acts on ERBB3 by adsorbing miR-145, thereby promoting the proliferation, migration, and invasion of HER2-positive breast cancer cells. More importantly, since ERBB3 belongs to the ErbB family, there have been a large number of studies to explore the treatment mechanism and efficacy of ERBB3 targeting HER2-positive breast cancer, and our study complements the mechanism of the occurrence and development of HER2-positive breast cancer and provides possible effective markers and treatment ideas.
Data Availability e data are available from the corresponding author Xing Li upon request via email (lixing@sysucc.org.cn).

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Yue Zhang and Duxun Tan have equally contributed to this study.

Supplementary Materials
e primer sequences for qRT-PCR and sirna sequences used in this study are provided in supplementary file. (Supplementary Materials)


s2fieldsofstudy: Biology Medicine Chemistry

text:
USP9X Increased Tumor Angiogenesis in Mantle Cell Lymphoma by Upregulation of CCND1-Mediated SOX11

Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy with a poor prognosis. Ubiquitin-specific peptidase 9, X-linked (USP9X), has been associated with multiple physiological pathways and regulates various cellular activities. In this study, we explored the role of USP9X in MCL in vitro and in vivo. USP9X was verified to be increased in peripheral blood mononuclear cells (PBMCs) of MCL patients and MCL cells. Moreover, CCND1 and SOX11 were also upregulated in PBMCs of MCL patients. The positive correlation between USP9X and CCND1, USP9X and SOX11, and CCND1 and SOX11 were identified. Further, USP9X overexpression and knockdown were performed in MCL cells. We proved that USP9X overexpression promoted proliferation and cell cycle and suppressed cell apoptosis in MCL cells. Upregulation of angiogenesis and cell migration were induced by USP9X overexpression in MCL cells. However, the USP9X knockdown showed opposite effects. In addition, USP9X was discovered to decrease Cyclin D1 (CCND1)-mediated SOX11 expression in MCL cells. We demonstrated that SOX11 overexpression reversed USP9X knockdown-mediated angiogenesis in MCL cells. Besides, tumor formation was inhibited by USP9X knockdown in mice in vivo. In conclusion, these results revealed that USP9X promoted tumor angiogenesis in MCL via increasing CCND1-mediated SOX11.

Introduction.
Mantle cell lymphoma (MCL) is a relatively rare subtype of highly aggressive B-cell lymphoma with approximately 7% of the non-Hodgkin lymphoma incidence rate. 1 Pathologically, it is featured by the chromosomal translocation t (11;14) (q13; q32). 2 MCL has a poor prognosis compared with other types of non-Hodgkin lymphoma, with a median survival time of only 3-5 years. 3 When most patients are diagnosed with stage III-IV, tumor infiltration usually occurs in lymph nodes, bone marrow, spleen, peripheral blood, and digestive tract. 4,5 In MCL, tumor cells can shape their microenvironment and activate a complex network of chemokines to promote tumor progression. For example, tumor stroma can increase endothelial cell proliferation and microvascular formation and enhance the aggressiveness of MCL. 6 Although a series of novel treatment approaches are applied to the improvement of survival rates of MCL patients, 7 about half of patients still suffer from recurrent MCL within 10 years. 8 Thus, treatment targets of MCL are necessary to be further explored.
Ubiquitin-specific peptidase 9, X-linked (USP9X), acts as a member of the USP family to be involved in multiple physiological pathways and regulate a variety of cellular activities. [9][10][11] For example, USP9X was highly expressed in pancreatic cancer, promoting epithelial mesenchymal transformation and inhibiting cell apoptosis. 12,13 Moreover, USP9X was associated with regulating tumor microenvironment and angiogenesis in breast cancer. 14 Additionally, it has been reported that USP9X expression was high in human invasive B-cell lymphoma samples and enhanced the chemoresistance of invasive B-cell lymphoma. 15 However, the underlying mechanism function of USP9X in MCL is still unclear.
CCND1, as a cell cycle regulatory protein, is located in chromosome 11q13 and directly regulated by p53. [16][17][18] Its upregulation was closely related to more advanced tumors and tumor metastasis. 19,20 Furthermore, CCND1 participated in regulating the malignant progression of MCL. 21 USP9X was proved to upregulate the expression of cell cycle-related protein CCND1, 22 suggesting that USP9X might function in MCL through CCND1.
Here, this study aims to investigate the effects of USP9X on the development of MCL. In addition, we confirmed the specific mechanism of USP9X on MCL through CCND1 in MCL, which provides a potential therapeutic target for MCL. Plasmids. For USP9X, CCND1, or SOX11 overexpression, human USP9X, CCND1, or SOX11 cDNA was cloned into the vector pcDNA3.0, respectively. USP9X or CCND1 knockdowns were purchased from JTSBIO (Wuhan, China). The USP9X or CCND1 shRNA target sequences and scrambled sequences were inserted into the expression siRNA vector-pRNAH1.1 vector.

Materials and Methods
Quantitative real-time PCR (qRT-PCR). Total RNA from peripheral blood mononuclear cells of MCL patients or MCL cells was extracted using TRIzol (Takara, Dalian, China). Then, cDNA was synthesized through universal cDNA synthesis kit II (Applied Biosystems, Foster City, CA, USA). Relative expression of USP9X was detected and calculated using the 2 −ΔΔCT method. Primers were used for this study as follows: Western blotting. Samples were lysed with RIPA lysis buffer. Proteins were extracted and subjected to SDSpolyacrylamide gels. Membranes were blocked with 5% skimmed milk and then incubated with primary antibodies, such as USP9X (1:1000) and β-actin (1:3000) (all from Abcam, Cambridge, MA, USA) at 4°C overnight. The corresponding HRP-conjugated secondary antibody was used to cover membranes. Bands were visualized using enhanced chemiluminescence substrate (Takara, Dalian, China).
Cell viability assay. The cell viability was measured through a Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China). Cells were seeded into 96 plates, and cell viability was examined at different periods (0, 24, 48, 72h). The OD value was detected using an MRX II microplate reader at 450 nm.
BrdU assay. Cells were fixed with 4% paraformaldehyde and then cut into 5 µm sections. Sections were deparaffinized and rehydrated and stained with BrdU. The images were captured under a light microscope.
Cell cycle analysis. Cells were collected and resuspended through pre-cooled 75% ethanol. Then, 500ul PBS containing 20ul RNAseA was added to cells for 30 min. Finally, cells were stained with PI (50 ug/mL) for 25 minutes. Flow cytometry was used for cell cycle analysis.
Cell apoptosis assay. Cells were collected and resuspended with 100 μL of binding buffer. Then, cells were stained with Annexin V-FITC and PI for 15 min. Finally, cell apoptosis was analyzed through a flow cytometer.
Transwell assay. For cell invasion analysis, Transwell chambers were added with Matrigel. For cell migration analysis, Transwell chambers were not added with Matrigel. Briefly, 4×10 5 cells were collected and resuspended with an FBS-free medium. Then, 200 μL of suspension was added to the top chamber and 500 μL of medium supplemented with 10% FBS was added to that lower. The cells were incubated for 48 h at 37°C, and cells were fixed and stained with 0.1% crystal violet. The images were observed under a microscope.
Animals. BALB/c nude mice (~4-5 weeks old) were divided into two groups (n=6/group): shNC; shUSP9X-2#. Then, 1 × 10 6 Z-138 cells with USP9X knockdown (shUSP9X-2#) and control cells (shNC) were subcutaneously injected into mice. Tumor growth was monitored for 30 days. Last, tumor tissues were collected and photographed when mice were sacrificed. According to Guide for the Care and Use of Laboratory Animals and (Yuebei people's Hospital), all experiments were conducted in.
Tube formation assay. Matrigel (50 microliters) was plated in 96-well plates. Human umbilical vein endothelial cells (HUVECs) were grown on Matrigel with different treatments and then incubated at 37°C for four h. Finally, tube formation was observed using microscopy and the branch points were calculated.
Immunohistochemistry and microvessel density (MVD) analysis. Tissues were fixed with 4% paraformaldehyde, and then paraffin embedding was conducted. Paraffinembedded tissues were cut into 5-µm-thick slides. After blocking with 1% BSA, the samples were incubated with primary antibodies, including TUNEL (1:1000), Ki67 (1:1500), USP9X (1:1000), CCND1 (1:1000), SOX11 (1:1000), and CD31 (1:1000) (all from Abcam, Cambridge, MA, USA) for 60 min at 37°C and then covered with goat anti-mouse secondary antibody for 50 min. Cell nuclei were stained with DAPI. Images were acquired under microscopy. Additionally, CD31positive MVD areas were calculated as the sum of areas of MVD (μm 2 ) evaluated divided by the total size of the core analyzed (μm 2 ) as previously described. 23 Statistical analysis. Experimental statistical analyses were performed using Measurement data were presented as mean ± SD. Two groups were compared with unpaired t-tests, and comparisons among multiple groups were conducted using ANOVA by Turkey. P less than 0.5 was considered a statistical significance.

Results.
USP9X was increased in PBMCs of MCL patients. The expression of USP9X, CCND1 and SOX11 in PBMCs of MCL patients was measured by qRT-PCR and western blotting. The results showed that USP9X, CCND1, and SOX11 mRNA levels and protein levels were high in peripheral blood mononuclear cells of MCL patients compared to the normal sample (Figure 1A, B). Moreover, a positive correlation between USP9X and CCND1, USP9X and SOX11, and CCND1 and SOX11 was proved by qRT-PCR, respectively ( Figure 1C). Additionally, qRT-PCR and western blotting demonstrated that USP9X expression was enhanced in MCL cell lines (Z-138, Mino, REC-1, Jeko-1, or JVM2) ( Figure 1E). These findings suggested that USP9X was increased in PBMCs of MCL patients and MCL cells.
USP9X promoted proliferation and cell cycle in MCL cells. The effect of USP9X on proliferation and cell cycle was detected in MCL cells by inducing either the USP9X overexpression either knockdown. Western blotting showed that USP9X overexpression increased USP9X protein level, and USP9X knockdown decreased USP9X protein level in Z-138 and Jeko-1 (Figure 2A). CCK-8 assay revealed that cell proliferation was promoted by USP9X overexpression, whereas it was inhibited by USP9X knockdown in Z-138 and Jeko-1 ( Figure 2B). Accordingly, USP9X overexpression elevated BrdU positive cells and USP9X knockdown reduced BrdU positive cells ( Figure 2C). Further, flow cytometry demonstrated that USP9X overexpression inhibited the G1 stage and promoted the S stage, whereas USP9X knockdown exerted opposite effects ( Figure 2D). Furthermore, western blotting verified that USP9X overexpression increased CDK4, CDK6, and PCNA protein levels and decreased P21 protein level. However, USP9X knockdown reversed these protein levels ( Figure 2E). These results indicated that USP9X promoted proliferation and cell cycle in MCL cells.
USP9X inhibited cell apoptosis in MCL cells. The effect of USP9X on cell apoptosis was explored in MCL cells. The results from flow cytometry was proved that USP9X overexpression suppressed cell apoptosis, and USP9X knockdown enhanced cell apoptosis in MCL cells (Z-138 and Jeko-1) (Figure 3A). Western blotting demonstrated that USP9X overexpression upregulated Bcl-2 protein levels and downregulated Bax and Cleaved caspase 3 protein levels. Conversely, USP9X knockdown showed opposite effects on these protein levels ( Figure 3B). The data implied that USP9X inhibited cell apoptosis in MCL cells.
USP9X contributed to angiogenesis and promoted cell migration in MCL cells. The role of USP9X in angiogenesis and cell migration was investigated. We found that tube formation was increased in HUVECs treated by USP9X overexpression-positive cell medium (CM), and it was decreased in HUVECs exposed to USP9X knockdown-positive CM (Figure 4A). Western blotting verified that USP9X overexpression elevated ANGPT2, FGF1, PDGFA, and VEGF protein levels, whereas USP9X knockdown repressed these protein levels in MCL cells (Z-138 and Jeko-1) (Figure 4B). In addition, cell migration and invasion were promoted by USP9X overexpression. However, USP9X knockdown inhibited cell migration and invasion in Z-138 and Jeko-1 (Figure 4C, D). BrdU revealed that USP9X knockdown decreased cell proliferation. CCND1 overexpression increased USP9X knockdown-caused cell proliferation and CCND1 T286A further promoted cell proliferation (Supplement Figure 1A). Flow cytometry proved that the cell cycle inhibited by USP9X knockdown was enhanced by CCND1 overexpression. In addition, CCND1 T286A further promoted the cell cycle (Supplement Figure 1B). These results indicated that USP9X contributed to angiogenesis and enabled cells to migrate in MCL tissue.

USP9X knockdown inhibited tumor formation in mice.
The effect of USP9X knockdown on tumor formation was explored in mice. Figure 7A and B shows that USP9X knockdown suppressed tumor volume and weight. IHC showed that USP9X knockdown increased TUNEL expression and decreased Ki67, USP9X, CCND1, and SOX11 expression in tumor tissues ( Figure 7C). Further, MVD revealed that USP9X knockdown repressed CD31 protein level using IHC ( Figure 7D). The data suggested that USP9X knockdown inhibited tumor formation in mice.

Discussion.
Here, USP9X, CCND1, and SOX11 were upregulated in PBMCs of MCL patients. USP9X level was increased in MCL cells. The positive correlation between USP9X and CCND1, USP9X and SOX11, and CCND1 and SOX11 were demonstrated. USP9X was identified to promote proliferation and cell cycle and inhibited cell apoptosis in MCL cells. Moreover, we found that USP9X contributed to angiogenesis and increased cell migration in MCL cells. Further, USP9X downregulated CCND1-mediated SOX11 expression in MCL cells. SOX11 overexpression was verified to reverse USP9X knockdown-mediated angiogenesis in MCL cells. In vivo, the results demonstrated that USP9X knockdown suppressed tumor formation in mice.
Previous studies have revealed that USP9X could target several cytosolic proteins and then be involved in regulating multiple cellular activities, such as cell apoptosis, cell growth, and migration. [24][25][26] The physical association of USP9X with centrosomes was dependent on the cell cycle and mostly measured in S and G2 phases. 27 Accordingly, our study demonstrated that USP9X increased proliferation and cell cycle in MCL cells. Furthermore, cell apoptosis was repressed by USP9X in MCL cells, evidenced by the upregulation of Bcl-2 protein level and downregulation of Bax and Cleaved caspase 3 protein levels. In addition, USP9X was also verified to contribute to angiogenesis and enhanced cell migration in MCL cells. These findings suggest that USP9X exerts a key role in MCL cells.
A recent study showed that the gain-of-function of   30 The relationship between angiogenesis and SOX11 expression has been revealed, and SOX11 was demonstrated to elevate an angiogenic phenotype in primary MCL. 31 Interestingly, our research verified that CCND1 knockdown repressed USP9X overexpressionmediated SOX11 protein level, suggesting that USP9X downregulated CCND1-mediated SOX11 expression in MCL cells. USP9X overexpression could promote WP1130-mediated USP9X, CCND1, and SOX11 protein levels, indicating that USP9X exerts its function by deubiquitinating Cyclin D1 and/or SOX11. Additionally, SOX11 overexpression was also found to reverse USP9X knockdown-mediated angiogenesis in MCL cells. Further analysis showed that USP9X knockdown could play a suppressive role in tumor formation in mice. USP9X knockdown reduced tumor volume and weight, inhibited TUNEL expression, and repressed Ki67, USP9X, CCND1, and SOX11 expression in tumor tissues. The data indicated that USP9X affected tumor angiogenesis in MCL through regulation of CCND1mediated SOX11. However, the mechanism of USP9X functions in MCL needs to be further explored. Additionally, the USP9X application in the clinical sample will be evaluated. Thus, more experiments will be conducted in the near future. In conclusion, we demonstrated that USP9X contributed to tumor angiogenesis. Furthermore, USP9X functioned in MCL through upregulating CCND1-mediated SOX11, which provides a new target for MCL patients.
Ethics approval. All procedures performed in studies involving human participants were in accordance with the standards upheld by the Ethics Committee of Yuebei people's Hospital and with those of the 1964 Helsinki Declaration and its later amendments for ethical research involving human subjects.
The Ethics Committee approved all animal experiments of Yuebei people's Hospital for the use of animals and conducted in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines.
Statement of Informed Consent. Written informed consent was obtained from a legally authorized representative(s) for anonymized patient information to be published in this article.
Authors' contributions. Gang Huang and Jianjun Liao designed the study, supervised the data collection, Mingli Wang analyzed the data, interpreted the data, and Yali Huang, Mingjie Tang, and Yanyan Hao prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript. References: